Project Summary/Abstract Staphylococcus aureus infections remain a major global health problem. In particular, antibiotic resistant staphylococcal infections remain a significant health burden to society. In the U.S. alone it is estimated that nearly five-hundred thousand cases of hospital-associated S. aureus infections occur yearly. These infections also contribute to pneumonia, sepsis, infective endocarditis, osteomyelitis and other diseases. S. aureus infections and associated diseases result from secreted virulence factors and the ability of the bacterium to survive in a wide-range of environmental niches, including hypoxic conditions. Importantly, growth and virulence are regulated by two-component systems (TCS). Two-component systems are generally composed of a membrane-bound sensor kinase and a cytoplasmic response regulator protein. The kinase senses the extracellular environment, and under the appropriate stimuli transmits a signal across the cell membrane to induce phosphorylation of the response regulator, resulting in changes in gene expression. The SrrAB TCS is critical for S. aureus survival under the hypoxic conditions at sites of infection and in the presence of oxidative and nitrosative stress. Moreover, the SrrAB TCS globally regulates the production of several virulence factors, including the toxic shock syndrome toxin-1 (TSST-1). In this proposal, the PI will pursue two aims designed to reveal the regulatory mechanisms of the SrrB sensor kinase in response to hypoxia and during oxidative and nitrosative stress. The first aim is to determine the biochemical mechanism(s) by which the SrrB enzyme senses and responds to the changes in redox state that occur under low-oxygen levels. The second aim is to determine the structural and dynamic bases for SrrB regulation using X-ray crystallography and small angle X- ray scattering. Together, these studies will have important implications for the regulation of SrrAB in pathogenicity and provide insight for the rational design of antimicrobials that target the SrrAB TCS.